US5846738A - Synthetic standard for immunoassays - Google Patents

Synthetic standard for immunoassays Download PDF

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US5846738A
US5846738A US08/762,695 US76269596A US5846738A US 5846738 A US5846738 A US 5846738A US 76269596 A US76269596 A US 76269596A US 5846738 A US5846738 A US 5846738A
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calibrator
peptidic
peptides
receptor
antigen
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Christoph Seidel
Peter Bialk
Herbert von der Eltz
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Roche Diagnostics GmbH
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Boehringer Mannheim GmbH
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4716—Muscle proteins, e.g. myosin, actin
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6887—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids from muscle, cartilage or connective tissue
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/86—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood coagulating time or factors, or their receptors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/96—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood or serum control standard
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G01N2333/4701—Details
    • G01N2333/4712—Muscle proteins, e.g. myosin, actin, protein
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/745—Assays involving non-enzymic blood coagulation factors
    • G01N2333/75—Fibrin; Fibrinogen
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/961—Chemistry: molecular biology and microbiology including a step of forming, releasing, or exposing the antigen or forming the hapten-immunogenic carrier complex or the antigen per se
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/967—Standards, controls, materials, e.g. validation studies, buffer systems
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/973—Simultaneous determination of more than one analyte
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S530/00—Chemistry: natural resins or derivatives; peptides or proteins; lignins or reaction products thereof
    • Y10S530/806—Antigenic peptides or proteins
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
    • Y10T436/00—Chemistry: analytical and immunological testing
    • Y10T436/10—Composition for standardization, calibration, simulation, stabilization, preparation or preservation; processes of use in preparation for chemical testing

Definitions

  • the invention concerns a peptidic synthetic standard that can be used in immunoassays.
  • the analytes to be determined in immunology are often human proteins such as e.g. creatine kinase (e.g. CKMB), HCG, fibrin monomer, prolactin or troponin T.
  • Human proteins isolated from natural sources are usually used to calibrate such tests. This isolation is difficult, expensive and requires the handling of human material that may be infectious. In this process it is also necessary to ensure that human proteins used for calibration contain no infectious contaminants.
  • the human proteins are not stable under all conditions. For example troponin T does not have an adequate long-term stability in solution as would be required for a liquid standard.
  • the object of the invention is to provide standards for immunological methods of determination for the detection of human proteins via binding of the analyte to at least one analyte-specific receptor which are stable, and simple and cheap to produce, and which do not have the aforementioned disadvantages.
  • a liquid stable calibrator for use in a test for the detection of an analyte comprising a conjugate of at least two binding sites that bind specifically to the analyte-specific binding region of the receptor that is used for detection in the test wherein the binding sites are connected by at least one soluble carrier substance and the conjugate is the product of a synthetic or recombinant production, dissolved in an aqueous solution in an exactly known amount.
  • the invention in addition concerns the use of this liquid stable calibrator to prepare a calibration curve in a test for the detection of an analyte.
  • step c) the result of the measurement from step b) is used to prepare a calibration curve
  • the test for the analyte in the sample is carried out and the analyte concentration is determined by comparing the measured signal with the calibration curve obtained from c).
  • a calibration curve is to be understood as the relation of a plurality of measured values to analyte concentrations.
  • conjugate is suitable in immunoassays as a synthetic standard and is stable over a long period in solution in wide pH ranges.
  • Conjugates that are each comprised of only one binding site that binds specifically to the analyte-specific region of the receptor that is used for detection in the test are less suitable as standards in such tests and only have a low affinity for the receptors.
  • FIG. 1 shows a preferred tree-like structure of a conjugate of the invention.
  • II and III are also suitable substructures, T: spacer, optional; Q: antibody binding site(s).
  • FIGS. 2a and 2b show the peptides A (FIG. 2a) and B (FIG. 2b) used in example 3.
  • FIGS. 3a and 3b show the conjugates synthesized in example 8 (FIG. 3a) and 9 (FIG. 3b).
  • FIG. 4 shows the standard curves that were obtained according to example 10 using the synthetic FM conjugates II and III and a fibrin standard of the state of the art.
  • Receptors are understood as all common molecules known to a person skilled in the art which bind to the analyte to be detected i.e. for example antibodies or binding proteins. Those receptors are particularly preferably used which have at least two analyte-specific binding regions i.e. for example antibodies or their bivalent fragments (F(ab) 2 ). The presence of two analyte-specific binding regions in the receptor enables a two-armed binding to the calibrator according to the invention in which at least two binding sites for the receptor are present per conjugate which is why, in comparison to a conjugate that only has one binding site, the binding constant is increased by the avidity of the receptor to the conjugate by 1000 to 10,000-fold.
  • calibrators according to the invention is also advantageous for monoclonal receptors such as Fab' fragments. These multivalent compounds enable the binding of a large number of labelled receptors which leads to a considerably increased signal.
  • Solid-phase-bound monovalent receptors have properties comparable with solid-phase-bound multivalent receptors so that the peptide derivatives according to the invention are also advantageous for monovalent solid-phase-bound receptors.
  • a binding site is understood as a peptide whose sequence is part of the protein sequence of a protein antigen (analyte) and to which a receptor directed towards this protein specifically binds.
  • a binding site is also to be understood as peptides with amino acid sequences which have a specificity and/or affinity of binding to the receptor that is essentially equivalent to that of the aforementioned peptides.
  • These peptides can preferably be derived from the aforementioned peptides by substitution, deletion or insertion of individual amino acid residues.
  • a binding site is also to be understood as peptide derivatives of the aforementioned peptides in which one or several amino acids have been derivatized by a chemical reaction.
  • peptide derivatives according to the invention are in particular those molecules in which the backbone or/and reactive amino acid side groups, such as free amino groups, free carboxyl groups or/and free hydroxyl groups, have been derivatized.
  • Specific examples of derivatives of amino groups are sulfonic acid amides or carboxylic acid amides, thiourethane derivatives and ammonium salts for example hydrochlorides.
  • Examples of carboxyl group derivatives are salts, esters and amides.
  • hydroxyl group derivatives are O-acyl or O-alkyl derivatives.
  • peptide derivative also encompasses those peptides in which one or several amino acids are substituted by naturally occurring or non-naturally occuring amino acid homologues of the 20 "standard" amino acids.
  • homologues are 4-hydroxyproline, 5-hydroxylysine, 3-methylhistidine, homoserine, ornithine, ⁇ -alanine and 4-aminobutyric acid.
  • the peptide derivatives must have specificity or/and affinity of binding to the receptors that is essentially equivalent to that of the peptides from which they are derived.
  • a binding site is also to be understood as peptide-mimetic substances, denoted peptide-mimetics in the following, which have an essentially equivalent specificity or/and affinity of binding to the receptors as the aforementioned peptides or peptide derivatives.
  • Peptide-mimetics are compounds that can substitute peptides with regard to their interaction with the receptor and can have an increased stability in particular towards proteinases or peptidases compared with the native peptides. Methods for the production of peptide-mimetics are described by Giannis and Kolter, "Angew. Chem.” 105 (1993), 1303-1326 and Lee et al., Bull. Chem. Soc. Jpn. 66 (1993), 2006-2010.
  • the length of a receptor binding site is usually at least 4 amino acids.
  • the length is preferably between 4 and 20, 4 and 15 or particularly preferably 4 and 10 amino acids.
  • An analogous length or size of the molecule is required in the case of peptide-mimetics.
  • the following peptides are for example suitable as binding sites for the determination of troponin T:
  • the peptides according to SEQ ID NO. 1, 2, 3, 4 and/or SEQ ID NO. 6 are particularly suitable.
  • the entire DNA and amino acid sequence of troponin T is described in Mesnard, L. et al FEBS Letters 328, 1.2, (1993) 139-144.
  • peptides are for example suitable as binding sites for the determination of fibrin monomer:
  • the conjugates according to the invention are particularly suitable as standards for the determination of troponin T, HCG and CKMB and fibrin monomer in immunological sandwich tests. Immunological sandwich tests for HCG, CKMB and fibrin monomer are described for example in:
  • the conjugate that is suitable in immunoassays as a synthetic standard contains at least two binding sites in each case which bind specifically to the analyte-specific binding region of the receptor that is used for detection in the test.
  • the conjugate contains two identical or similar binding sites both of which bind to this receptor.
  • the conjugate in each case contains two identical or similar binding sites that are capable of binding to the two receptors.
  • the conjugate thus contains two binding sites for the first and two binding sites for the second receptor.
  • the conjugate contains at least four identical or similar binding sites which are capable of binding to the receptors.
  • the number of binding sites can, however, also be larger. If the conjugate contains a soluble polymer of high molecular weight as the carrier substance, up to 50 binding sites for each receptor binding region may be present. If the binding sites in the conjugate are coupled to the carrier substance to form a linear or branched synthetic oligomoer of amino acids and spacer molecules, then 2 to 16 is the preferred number of binding sites.
  • the carrier substance is preferably composed of a plurality of peptidic oligomers that are arranged between the binding sites. In such cases the carrier substance is preferably identical to the spacer.
  • Branched conjugates preferably have a tree-like structure (cf. e.g. FIG. 1). The number of binding sites in such conjugates is preferably 2, 4, 8 or 16. In this case it is preferable that spacers are inserted between the ends of the carrier substance and the binding sites.
  • the binding sites are covalently linked together via branching points with 2, 4 or 8 amino groups as the carrier substance.
  • branching point is a bifunctional or multifunctional molecule on the functionalities of which binding sites or, if necessary, further branching points can be bound. This binding can be direct or via a spacer.
  • Preferred branching points are for example ⁇ , -bisamino acids such as lysine or ⁇ -bisamines.
  • Such branched peptides are for example described in Tam, J. P. Proc. Natl. Acad. Sci. USA 85 (1988) 5409-5431, WO 92/18528 and Marsden, H. S. et al., J. Immunol. Meth. 147 (1992) 65-72.
  • a branching site is understood as a chemical group that can bind to a chemical group in a binding site (if desired after prior activation). Examples of this are primary or secondary amino, carboxylic acid, sulfhydryl, alcohol, aldehyde and sulfonic acid functional groups.
  • Lysine and homooligomers of lysine preferably with 3 to 7 lysines are also preferred as the carrier substance for conjugates with a tree-like structure.
  • binding sites which in each case are directed towards the same receptor can either have an identical or different amino acid sequence. However, it must be ensured that the binding sites specifically recognize the same binding region of the receptors.
  • a spacer is understood as a bifunctional molecule that can bind to the ends of two binding sites or to one binding site and to the soluble carrier and as a result produces a spatial distance between these binding sites.
  • the spacers are preferably linear molecules with adequate polar properties for water solubility.
  • the spacer is also particularly preferably composed of amino acid residues.
  • These amino acids can correspond to the natural intermediate sequence of the binding sites in the natural analyte or be different from the natural sequence.
  • natural amino acids such as for example alanine, glycine, cysteine, lysine, aspartic acid, glutamic acid, serine, leucine or non-natural linear ⁇ -amino acids such as e.g. ⁇ -alanine, ⁇ -amino butyric acid or ⁇ -aminocaproic acid are again preferred. They are coupled via covalent bonds (e.g.
  • spacer length must be chosen such that the receptors can bind substantially unhindered to the binding sites.
  • a spacer length of over 40 atoms does not have any further particular advantages. In addition such molecules are more time-consuming to synthesize.
  • the spacers are preferably composed of 1-10 amino acids in each case.
  • Preferred spacers are: oligoglycines, oligo- ⁇ -alanines, oligo- ⁇ -aminocaproic acids or oligopeptides made of ⁇ -alamines and ⁇ -aminocaproic acids.
  • the N-terminus of the conjugate can either be free or contain a bound organic residue such as e.g. an acetyl, tertiary butyloxycarboxyl, peptidyl, aryl or alkyl residue.
  • the C-terminus can also be present in a free form as a carboxylate or amide or it can be derivatized with an organic residue and for example be an alkyl ester or an aryl ester.
  • the C-terminus and N-terminus are linked via a bivalent spacer or a peptide sequence so that the molecule has a cyclic structure.
  • the carrier substance can be a combination of spacer and branching points and represent a polymer which has a plurality of functional binding groups for the binding sites.
  • polymers can for example be soluble polyamines, dextrans, polyacrylates, polymethacrylates, polypeptides, proteins or nucleic acids with a molecular weight of 1000 to 5 million Daltons, but preferably of 10,000 to 1 million Daltons. Soluble proteins such as albumins, immunoglobulins or ⁇ -galactosidase are particularly suitable as polymers.
  • the polymers carry 2 to 500 groups capable of coupling. A large number of suitable methods for producing the bond between the binding sites and polymers is known to a person skilled in the art.
  • the binding can for example be carried out via hetero of homobifunctional linkers or via the direct formation of an amide bond. It is particularly expedient to react a polymer carrying amino groups with a N-hydroxysuccinimide ester derivative of the binding site.
  • the direct reaction of a polymer carrying SH groups with maleinimide derivatives of the binding site or the use of maleinimide-derivatized carrier polymers and binding sites carrying SH groups is particularly suitable.
  • the conjugates used according to the invention can be prepared according to known methods such as those described for example by J. P. Tam, Proc. Natl. Acad. Sci. USA, 58 (1988), 5409-5413; D. N. Pressmett et al., J. Biol. Chem. 263 (1988), 1719-1725; H. Lankinen, J. Gen. Virol. 70 (1989), 3159-3169; WO 92/18528 or H. S. Marsden, J. Immunol. Methods 147 (1992), 65-72.
  • the conjugates according to the invention comprising at least two binding sites and at least one soluble carrier substance are used as liquid calibrators to prepare a calibration curve in a test for the detection of an analyte.
  • liquid calibrators When preparing the liquid calibrators according to the invention one must take care that the conjugates are present in an exactly defined and known amount in the liquid calibrator. It is expedient to provide the liquid calibrators in various concentrations in order to prepare a calibration curve so that no further dilution steps are necessary before use.
  • the concentration of the conjugate in the liquid calibrator depends on the analyte to be detected and usually covers the analyte concentrations that occur physiologically. Calibrator series are provided in typical concentrations of 0.1 to 1000 ng/ml.
  • a liquid calibrator series for the troponin T test covers for example the conjugate concentrations of 0, 0.1, 0.5, 1, 5, 10, 50, 100 and 500 ng/ml.
  • the conjugate concentration in the calibrator series is for example 10, 50, 100, 250 and 500 ng/ml.
  • liquid stable calibrator means that after dissolving an exactly defined amount of the conjugate in a particular amount of a solvent, usually a buffer, the liquid calibrator which is thereby formed has a long stability.
  • the dissolution of the conjugate in the solvent can already be carried out by the manufacturer. This has the advantage that the end user does not have to carry out any additional working steps.
  • the conjugate can also be dissolved by the user.
  • the conjugate is provided by the manufacturer as a dry substance, for example as a lyophilisate. This has the advantage that an improper transport or an improper storage before use i.e. dissolution is less damaging.
  • the liquid calibrator contains further auxiliary substances such as for example buffers, stabilizers or preservatives in an exactly defined and known amount in addition to the conjugate.
  • Sodium phosphate, MES or PIPES have proven to be particularly suitable as the buffer.
  • the pH value is preferably 5.0-6.0.
  • Bovine serum albumin can for example be used as the stabilizers.
  • the calibrators according to the invention have a higher stability on storage than stabilizers that have been produced from the naturally occurring analytes. This effect is particularly pronounced when the analyte is a labile protein such as troponin T. The reasons for this can for example be proteolytic attack, unspecific binding to surfaces or a tendency to aggregate.
  • the invention in addition concerns a liquid stable universal calibrator for use in several tests for the detection of at least two different analytes.
  • the universal calibrator is composed of a mixture of at least two different aforementioned calibrators. Since these synthetic calibrators are exactly defined substances, their mutual influence in the mixture is very much smaller than with material from an organic source.
  • the conjugates according to the invention can be used in homogeneous immunoassays as well as in heterogeneous immunoassays.
  • Homogeneous assays are for example understood as a precipitation method such as TINIA (turbidimetric inhibition assay), an agglutination method, FPIA (fluorescence polarisation immunoassay) or CEDIA (cloned enzyme donor immunoassay) i.e. methods in which the degree of receptor-analyte reaction is quantified without separating the free and bound fractions.
  • Heterogeneous assays are for example understood as radioimmunoassays or sandwich assays i.e.
  • Enzymatic label or fluorescent labels are for example used as the label in sandwich immunoassays. Such labels are known to a person skilled in the art and are described for example in:
  • the conjugates according to the invention have proven to be particularly suitable for use in sandwich assays.
  • the determination is carried out by incubating the sample with at least two analyte-specific receptors wherein one of these receptors is immobilized in a solid phase before or after the reaction with the analyte and the second receptor carries a label.
  • the liquid phase and solid phase are separated and the label is determined in one of the two phases as a measure of the amount of analyte.
  • a known amount of the conjugate according to the invention is bound instead of the sample to the analyte-specific receptor.
  • a calibration curve is prepared from these results. Afterwards the analyte test is carried out and the analyte concentration is determined by comparing the measured signal with the calibration curve.
  • the conjugate according to the invention must have a particular composition depending on the method. If a method of detection is used in which only one analyte-specific receptor is necessary i.e. for example a competitive method, then a conjugate of at least two binding sites linked by at least one soluble carrier substance is used.
  • a conjugate of at least two binding sites for each of these two receptors linked by at least one soluble carrier substance is used.
  • the two receptors can either be identical i.e. recognize the same epitopes of the antigen or they can be different i.e. recognize different epitopes of the antigen.
  • the antigen must have several identical epitopes which is for example the case for antigens with repetitive epitopes or identical subunits. An example of this is fibrin monomer.
  • the conjugate must therefore have at least four identical binding sites.
  • the conjugate contains at least two binding sites for the first receptor and at least two binding sites for the second different receptor. Troponin T can for example be detected in this manner.
  • FIG. 1 shows a preferred tree-like structure of a conjugate suitable according to the invention.
  • II and III are also suitable substructures, T: spacer, optional; Q: antibody binding site(s).
  • FIGS. 2a and 2b show the peptides A (FIG. 2a) and B (FIG. 2b) used in example 3.
  • FIGS. 3a and 3b show the conjugates synthesized in example 8 (FIG. 3a) and 9 (FIG. 3b).
  • FIG. 4 shows the standard curves that were obtained according to example 10 using the synthetic FM conjugates II and III and a fibrin standard of the state of the art.
  • the conjugate (I) was produced by means of Fmoc-(fluorenylmethoxy-carbonyl-)-solid phase peptide synthesis using a SMPS 350 peptide synthesizer from the ZINSSER Analytic Company on 15 mg 4-(2',4'-dimethoxy-phenyl-Fmoc-aminomethyl)-phenoxy resin SA5030 from the ADVANCED CHEMTECH Company with a loading of 0.22 mmol/g.
  • butyl ester arginine(-pentamethylchromane), glutamic acid(-tert. butyl ester), asparagine(-trityl), arginine(-pentamethylchromane), glutamic acid(-tert. butyl ester), alanine, arginine (-pentamethylchromane, arginine(-pentamethylchromane), N ⁇ -tert. butyloxycarbonyl-lysine, glutamic acid(-tert. butyl ester), isoleucine, arginine(-pentamethylchromane), aspartic acid(-tert. butyl ester), N ⁇ -tert.
  • the coupling times are 40 and 30 minutes.
  • the cleavage time of the Fmoc protecting group is carried out after each double coupling using 600 ⁇ l of a 50% solution of piperidine in dimethylformamide.
  • the cleavage time is 20 minutes.
  • the washing steps are carried out eight times after each of the reaction steps with 700 ⁇ l dimethyl-formamide each time.
  • the peptide is released by treating the resin filtered free of solvent with 750 ⁇ l in each case of a mixture of 90% trifluoroacetic acid, 3% thioanisole, 3% ethanedithiol and 3% thiocresol within 20 minutes and subsequently for 140 minutes.
  • the conjugate (II) was produced by means of Fmoc-(fluorenylmethoxy-carbonyl-)-solid phase peptide synthesis using a SMPS 350 peptide synthesizer from the ZINSSER Analytic Company on 15 mg 4-(2',4'-dimethoxy-phenyl-Fmoc-aminomethyl)-phenoxy resin SA5030 from the ADVANCED CHEMTECH Company with a loading of 0.22 mmol/g.
  • butyl ester asparagine (trityl), arginine (pentamethylchromane), isoleucine, arginine (pentamethylchromane), glutamine (trityl), glutamine (trityl), glutamic acid (tert. butyl ester), alanine, arginine (pentamethylchromane), glutamic acid (tert. butyl ester), alanine, arginine (pentamethylchromane), glutamic acid (tert. butyl ester), alanine, arginine (pentamethylchromane), arginine (pentamethylchromane), lysine (tert. butyloxycarbonyl), glutamic acid (tert.
  • the peptide is released by treating the resin filtered free of solvent with 750 ⁇ l in each case of a mixture of 90% trifluoroacetic acid, 3% thioanisole, 3% ethanedithiol and 3% thiocresol within 20 minutes and subsequently for 140 minutes.
  • the product is precipitated by addition of 15 ml cold diisopropyl ether to the pooled filtrate and isolated by filtration. The residue is dissolved in 3 ml 50% acetic acid and lyophilized. The lyophilisation procedure is repeated twice. 17 mg crude material with a purity of 52% according to reverse-phase HPLC is obtained of which 5 mg is purified by means of preparative reverse-phase HPLC. 1 mg pure material (according to HPLC >99%) is obtained.
  • a biotin-labelled monoclonal antibody (MAB) against troponin T and a ruthenium-Tris(bispyridyl)-labelled antibody are incubated for 10 minutes with troponin T-coated and streptavidin-coated magnetic polymer particles (beads). During the incubation period the immunocomplex as well as the binding of the biotinylated MABs to the magnetic particles is formed.
  • the reaction is ended by withdrawing the reaction mixture in the measuring cell by suction.
  • the magnetic polymer particles are immobilized on the electrode in the measuring chamber by a magnet.
  • the remaining reaction mixture is removed by suction and the magnetic particles are washed with assay buffer. Subsequently the light emission is excited by applying an electrical voltage.
  • BSA bovine serum albumin
  • Biotinylated MAB against troponin T 2 ⁇ g/ml in incubation buffer.
  • the conjugates to be tested are weighed in human serum or incubation buffer.
  • the signal which one obtains by measuring these samples in methods described above is compared with the signal which is obtained with the calibration solutions.
  • the quotient recovery/weighed-in quantity is calculated from this.
  • each of the solutions 1-4 is incubated with 60 ⁇ l sample for 10 minutes at room temperature. Afterwards the reaction mixture in the measuring chamber is removed by suction and the magnetic particles are separated. The measuring chamber is filled with assay buffer and the measured signal is recorded. The concentration of the samples is determined by comparing the signal with that of the calibration solutions.
  • the measurement was carried out on an Origen 1.0 instrument from the Igen Company USA.
  • bovine serum albumin 2.9 ⁇ mol
  • maleinimidohexanoic acid-N-hydroxysuccinimide ester 145 ⁇ mol is dissolved in 0.5 ml dioxan and added dropwise to the above solution. It is allowed to stir for 17 hours at room temperature (RT).
  • the first peak is collected and its protein content is determined according to the method of Pierce (BCA bicinchoninic acid method).
  • the loading of maleinimidohexanoyl groups obtained is determined according to the method of Ellman. It is 17.5 maleinimidohexanoyl groups per molecule bovine serum albumin.
  • the solution is saturated with argon.
  • 2.13 ml of a solution of maleinimidohexanoyl-activated bovine serum albumin (loading 17.5:1) at a concentration of 9.46 mg/ml in 0.1M potassium phosphate buffer pH 6.0 is added to the above solution. It is again saturated with argon and stirred at room temperature for 17 hours while excluding oxygen.
  • 4 ml 0.05M succinic acid buffer pH 4.0 is added and it is dialysed 4 times against 1 liter 1% acetic acid. The insoluble precipitate is centrifuged.
  • bovine serum albumin (2.9 ⁇ mol) is dissolved in 6 ml 0.1M potassium phosphate buffer pH 8.0.
  • 44.6 mg maleinimidohexanoic acid-N-hydroxysuccinimide ester (145 ⁇ mol) is dissolved in 0.5 ml dioxan and added to the above solution. It is allowed to stir for 17 hours at room temperature.
  • the first peak is collected and its protein content is determined according to the method of Pierce (BCA method).
  • the loading of maleinimidohexanoyl groups obtained is determined according to the method of Ellman as 18.0 per molecule bovine serum albumin.
  • the stability of the troponin T conjugate (I) according to example 1 was compared with the stability of a calibrator made from native antigen.
  • Troponin T from bovine heart (bcTnT) and human heart (hcTnT) was used as the native antigen.
  • the calibrators were stored for up to three weeks at temperatures of -80° C., 4° C. and 25° C.
  • the measured signal obtained with this calibrator was determined at weekly intervals and compared with the initial signal.
  • the test procedure corresponds to example 3.
  • the superior stability of the calibrator according to the invention was apparent when stored at 25° C. Whereas the measured signal in the case of hcTnT and bcTnT fell to 24 and 5% respectively of the initial signal after 3 weeks at 25° C., the signal of the synthetic calibrator was still 50% of the initial value.
  • the FM conjugate (I) for the test for soluble fibrin was synthesized by means of Fmoc-(fluorenylmethoxycarbonyl-) solid phase peptide synthesis using a SMPS 350 peptide synthesizer from the ZINSSER Analytic Company on 15 mg 4-(2',4'-dimethoxyphenyl-Fmoc-aminomethyl)-phenoxy resin SA 5030 from the ADVANCED CHEMTECH Company with a loading of 0.22 mmol/g.
  • the cleavage time of the Fmoc protecting group is carried out after each double coupling with 600 ⁇ l of a 50% solution of piperidine in dimethylformamide.
  • the washing steps are carried out eight times after each of the reaction steps with 700 ⁇ l dimethylformamide each time.
  • the peptide is released by treating the resin filtered free of solvent with 750 ⁇ l in each case of a mixture of 90% trifluoroacetic acid, 3% thioanisole, 3% ethanedithiol and 3% thiocresol within 20 minutes and subsequently for 140 minutes.
  • the product is precipitated by addition of 15 ml cold diisopropyl ether to the pooled filtrate and isolated by filtration.
  • conjugate is formed of two peptides corresponding to the sequence shown in SEQ ID NO. 11 which are each linked to lysine via a spacer composed in each case of two molecules of ⁇ -alanine.
  • the FM conjugate (II) for the test for soluble fibrin was synthesized by means of Fmoc-(fluorenylmethoxycarbonyl-) solid phase peptide synthesis using a SMPS 350 peptide synthesizer from the ZINSSER Analytic Company on 15 mg 4-(2',4'-dimethoxyphenyl-Fmoc-aminomethyl)-phenoxy resin SA 5030 from the ADVANCED CHEMTECH Company with a loading of 0.22 mmol/g.
  • the coupling times are 40 and 30 minutes.
  • the cleavage time of the Fmoc protecting group is carried out after each double coupling using 600 ⁇ l of a 50% solution of piperidine in dimethylformamide.
  • the washing steps are carried out eight times after each of the reaction steps with 700 ⁇ l dimethylformamide each time.
  • the peptide is released by treating the resin filtered free of solvent with 750 ⁇ l in each case of a mixture of 90% trifluoroacetic acid, 3% thioanisole, 3% ethane-dithiol and 3% thiocresol within 20 minutes and subsequently for 140 minutes.
  • a conjugate is formed of four peptides corresponding to the sequence shown in SEQ ID NO. 11 which are each linked via a spacer of two molecules to a carrier composed of 3 lysine residues (FIG. 3a).
  • the FM conjugate (III) for a test for soluble fibrin was synthesized by means of Fmoc-(fluorenylmethoxycarbonyl-) solid phase peptide synthesis using a SMPS 350 peptide synthesizer from the ZINSSER Analytic Company on 15 mg 4-(2',4'-dimethoxyphenyl-Fmoc-aminomethyl)-phenoxy resin SA 5030 from the ADVANCED CHEMTECH Company with a loading of 0.22 mmol/g.
  • the coupling times are 40 and 30 minutes.
  • the cleavage time of the Fmoc protecting group is carried out after each double coupling using 600 ⁇ l of a 50% solution of piperidine in dimethylformamide.
  • the washing steps are carried out eight times after each of the reaction steps with 700 ⁇ l dimethylformamide each time.
  • the peptide is released by treating the resin filtered free of solvent with 750 ⁇ l in each case of a mixture of 90% trifluoroacetic acid, 3% thioanisole, 3% ethanedithiol and 3% thiocresol within 20 minutes and subsequently for 140 minutes.
  • a conjugate is formed of eight peptides corresponding to the sequence shown in SEQ ID NO. 11 which are each linked via a spacer of two ⁇ -alanine molecules to a carrier composed of 7 lysine residues (FIG. 3b).
  • a two-step sandwich assay was carried out using streptavidin-coated tubes as the solid phase.
  • the same fibrin-specific monoclonal antibody was used as the biotinylated capture MAB and as the peroxidase-labelled MAB.
  • the test was carried out at room temperature on an ES 33 instrument.
  • the peptide was coupled by means of maleinimidobenzoyl-N-hydroxysuccinimide ester to Keyhole limpet haemocyanin.
  • the monoclonal antibodies were obtained according to Kohler and Milstein, Nature 256 (1975), 495-497.
  • the tube was subsequently washed with 4.3 mM NaCl solution.
  • 1000 ⁇ l peroxidase-labelled antibody solution (0.14 U/ml peroxidase conjugate, 0.035M potassium phosphate, 0.154M NaCl, 10 mg/ml polyethylene glycol 40000, 2 mg/ml bovine serum albumin, 0.5 mg/ml Tween 20®, pH 7.4) was added and it was incubated for 30 minutes. Afterwards the solution was removed from the tube and this was washed with 4.3 mM NaCl solution.
  • FIG. 4 shows standard curves. These were determined in the range of 0 to 50 ⁇ g/ml fibrin.

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Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6190916B1 (en) * 1999-06-29 2001-02-20 Spectral Diagnostics, Inc. Troponin I composition
US20030173283A1 (en) * 1998-08-17 2003-09-18 Pulek John L. Graded particle-size retention filter medium for fluid filtration unit with improved edge seal
US6867011B1 (en) * 1996-12-05 2005-03-15 Bio-Rad Pasteur Synthetic bioepitope compounds which can be used as standards in the biological assays of Troponin I
WO2007103734A2 (en) * 2006-03-03 2007-09-13 Ciphergen Biosystems, Inc. Method for quantifying an analyte in a biological sample using mass spectrometry
US20080032281A1 (en) * 2004-06-01 2008-02-07 Umedik Inc. Method and Device for Rapid Detection and Quantitation of Macro and Micro Matrices
US20080220980A1 (en) * 2004-07-20 2008-09-11 Umedik Inc. Method to Measure Dynamic Internal Calibration True Dose Response Curves
US20080269075A1 (en) * 2004-07-20 2008-10-30 Umedik Inc. Method and Device to Optimize Analyte and Antibody Substrate Binding by Least Energy Adsorption
US7479278B2 (en) 1998-10-21 2009-01-20 Spectral Diagnostics, Inc Troponin I polypeptide fragments and uses thereof
US20110076697A1 (en) * 2009-04-28 2011-03-31 Innovative Laboratory Technologies, Inc. Lateral-flow immuno-chromatographic assay devices
US8013115B1 (en) * 1999-08-16 2011-09-06 Bio-Rad Pasteur Use of stabilised synthetic compounds in immunoassay
US8546148B2 (en) 2010-06-14 2013-10-01 Siemens Healthcare Diagnostics Inc Composition for use as an assay reagent
CN105628941A (zh) * 2015-12-22 2016-06-01 北京康彻思坦生物技术有限公司 一种IgM血清学检测质控物的制备方法
US11193929B2 (en) * 2014-12-18 2021-12-07 Biomerieux Synthetic bi-epitope compound

Families Citing this family (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2734267B1 (fr) * 1995-05-16 1997-08-01 Pasteur Sanofi Diagnostics Composition stabilisee de troponine pour immunoessais et procede de preparation d'une telle composition stabilisee
DE19524572A1 (de) * 1995-07-06 1997-01-09 Bayer Ag Verfahren zur Herstellung eines synthetischen Kalibrators für den Einsatz in Immunoassays, bestehend aus den Analyten oder Teilsequenzen davon, die an inerten Trägermoleküle konjugiert sind
DE19548028A1 (de) * 1995-12-21 1997-06-26 Bayer Ag Verfahren zur Herstellung eines synthetischen Kalibrators für den Einsatz in Sandwich-Immunoassays, bestehend aus einem Antikörper gegen einen der im Assay benutzten Antikörper und einer Sequenz des Analyten
DE19637803A1 (de) * 1996-09-17 1998-03-19 Boehringer Mannheim Gmbh Stabilisierung von Standardlösungen durch Konjugate
EP0946198B1 (de) * 1996-10-15 2002-06-19 Medical Analysis Systems, Inc. Verfahren zur Stabilisierung von Troponin I (CTnI) durch Konjugation mit einem activen Polymer

Citations (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE3112334A1 (de) * 1981-03-28 1982-10-07 Behringwerke Ag, 3550 Marburg "kuenstliche kontroll- und standardseren, verfahren zu ihrer herstellung und ihre verwendung"
EP0306813A2 (de) * 1987-09-08 1989-03-15 BEHRINGWERKE Aktiengesellschaft Verfahren zur Bestimmung von "löslichem" Fibrin
US4859765A (en) * 1983-10-17 1989-08-22 Syntex (U.S.A.) Inc. Synthetic peptide sequences useful in biological and pharmaceutical applications and methods of manufacture
US4886663A (en) * 1983-01-03 1989-12-12 Scripps Clinic And Research Foundation Synthetic heat-stable enterotoxin polypeptide of Escherichia coli and multimers thereof
US4939094A (en) * 1985-08-28 1990-07-03 Kyowa Hakko Kogyo Co., Ltd. Fused antigen polypeptide
EP0394819A2 (de) * 1989-04-25 1990-10-31 Roche Diagnostics GmbH Spezifische Antikörper gegen Troponin T, ihre Herstellung und Verwendung in einem Reagenz zur Bestimmung von Herzmuskelnekrosen
JPH03200065A (ja) * 1989-12-28 1991-09-02 Tosoh Corp 免疫測定用参照品及びその製造方法
EP0472205A1 (de) * 1990-08-23 1992-02-26 New York Blood Center, Inc. Testverfahren, die ein lösliches fibrinähnliches Monomer nutzen
US5357042A (en) * 1984-04-23 1994-10-18 The General Hospital Corporation Synthetic peptides capable of eliciting fibrin-specific monoclonal antibodies lacking fibrinogen-cross-reactivity
US5411869A (en) * 1992-02-04 1995-05-02 Ohmicron Corporation Immunological analogs for captan
US5436126A (en) * 1990-02-16 1995-07-25 United Biomedical, Inc. Synthetic peptides specific for the detection of antibodies to HCV, diagnosis of HCV infection and prevention thereof as vaccines
US5639854A (en) * 1993-06-09 1997-06-17 Connaught Laboratories Limited Tandem synthetic HIV-1 peptides

Family Cites Families (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4271140A (en) * 1978-01-23 1981-06-02 Baxter Travenol Laboratories, Inc. Method and composition for double receptor, specific binding assays
DE2965570D1 (en) * 1978-08-24 1983-07-07 Akzo Nv Method for the detection and/or determination of an antigen specific immunoglobulin, immuno-reagents to be used in said method and test kit for said determination
DE3400434A1 (de) * 1984-01-09 1985-09-26 Behringwerke Ag, 3550 Marburg Bestimmung von fibrin mit fibrin-spezifischen antikoerpern
US5206086A (en) † 1987-10-13 1993-04-27 University Of Rochester Particle-stabilized epitopes for standardization and control of immunoassays
EP0339695A3 (de) * 1988-04-29 1990-05-30 Stichting Voor De Technische Wetenschappen Verfahren zur Herstellung eines antigenen bzw. immunogenen Konjugats sowie die Verwendung solcher Konjugate
DE3822750A1 (de) * 1988-07-05 1990-01-11 Boehringer Mannheim Gmbh Verfahren zur bestimmung einer spezifisch bindefaehigen substanz
US5223393A (en) * 1990-06-12 1993-06-29 Microgenics Corporation Detection of analytes having binding sites for at least two binding moieties

Patent Citations (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE3112334A1 (de) * 1981-03-28 1982-10-07 Behringwerke Ag, 3550 Marburg "kuenstliche kontroll- und standardseren, verfahren zu ihrer herstellung und ihre verwendung"
US4886663A (en) * 1983-01-03 1989-12-12 Scripps Clinic And Research Foundation Synthetic heat-stable enterotoxin polypeptide of Escherichia coli and multimers thereof
US4859765A (en) * 1983-10-17 1989-08-22 Syntex (U.S.A.) Inc. Synthetic peptide sequences useful in biological and pharmaceutical applications and methods of manufacture
US5357042A (en) * 1984-04-23 1994-10-18 The General Hospital Corporation Synthetic peptides capable of eliciting fibrin-specific monoclonal antibodies lacking fibrinogen-cross-reactivity
US4939094A (en) * 1985-08-28 1990-07-03 Kyowa Hakko Kogyo Co., Ltd. Fused antigen polypeptide
EP0306813A2 (de) * 1987-09-08 1989-03-15 BEHRINGWERKE Aktiengesellschaft Verfahren zur Bestimmung von "löslichem" Fibrin
EP0394819A2 (de) * 1989-04-25 1990-10-31 Roche Diagnostics GmbH Spezifische Antikörper gegen Troponin T, ihre Herstellung und Verwendung in einem Reagenz zur Bestimmung von Herzmuskelnekrosen
JPH03200065A (ja) * 1989-12-28 1991-09-02 Tosoh Corp 免疫測定用参照品及びその製造方法
US5436126A (en) * 1990-02-16 1995-07-25 United Biomedical, Inc. Synthetic peptides specific for the detection of antibodies to HCV, diagnosis of HCV infection and prevention thereof as vaccines
EP0472205A1 (de) * 1990-08-23 1992-02-26 New York Blood Center, Inc. Testverfahren, die ein lösliches fibrinähnliches Monomer nutzen
US5411869A (en) * 1992-02-04 1995-05-02 Ohmicron Corporation Immunological analogs for captan
US5639854A (en) * 1993-06-09 1997-06-17 Connaught Laboratories Limited Tandem synthetic HIV-1 peptides

Non-Patent Citations (8)

* Cited by examiner, † Cited by third party
Title
Derwent Abstract of TOSOH Corp. 91 3000 11/41 JP403200065A, Sep. 1991. *
Derwent Abstract of TOSOH Corp. 91-3000 11/41 JP403200065A, Sep. 1991.
FG Gabriel et al., "Peptide based enzyme immunoassays for detecting hepatitis C antibodies . . . " J. Clin. Pathol. 47(4):357-359, Apr. 1994.
FG Gabriel et al., Peptide based enzyme immunoassays for detecting hepatitis C antibodies . . . J. Clin. Pathol. 47(4):357 359, Apr. 1994. *
H.S. Marsden et al., "Advantages of branched peptides in serodiagnosis," J. of Immunol. Methods 147:65-72, 1992.
H.S. Marsden et al., Advantages of branched peptides in serodiagnosis, J. of Immunol. Methods 147:65 72, 1992. *
L. Mesnard et al., "Molecular Cloning and developmental expression of human cardiac troponin T," FEBS Letters 328(1,2):139-144, Aug. 1993.
L. Mesnard et al., Molecular Cloning and developmental expression of human cardiac troponin T, FEBS Letters 328(1,2):139 144, Aug. 1993. *

Cited By (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6867011B1 (en) * 1996-12-05 2005-03-15 Bio-Rad Pasteur Synthetic bioepitope compounds which can be used as standards in the biological assays of Troponin I
US20030173283A1 (en) * 1998-08-17 2003-09-18 Pulek John L. Graded particle-size retention filter medium for fluid filtration unit with improved edge seal
US7479278B2 (en) 1998-10-21 2009-01-20 Spectral Diagnostics, Inc Troponin I polypeptide fragments and uses thereof
US6190916B1 (en) * 1999-06-29 2001-02-20 Spectral Diagnostics, Inc. Troponin I composition
US8013115B1 (en) * 1999-08-16 2011-09-06 Bio-Rad Pasteur Use of stabilised synthetic compounds in immunoassay
US20080032281A1 (en) * 2004-06-01 2008-02-07 Umedik Inc. Method and Device for Rapid Detection and Quantitation of Macro and Micro Matrices
US11422129B2 (en) 2004-07-20 2022-08-23 Sqi Diagnostics Systems Inc. Method and device to optimize analyte and antibody substrate binding by least energy adsorption
US20080220980A1 (en) * 2004-07-20 2008-09-11 Umedik Inc. Method to Measure Dynamic Internal Calibration True Dose Response Curves
US20080269075A1 (en) * 2004-07-20 2008-10-30 Umedik Inc. Method and Device to Optimize Analyte and Antibody Substrate Binding by Least Energy Adsorption
WO2007103734A3 (en) * 2006-03-03 2007-12-06 Ciphergen Biosystems Inc Method for quantifying an analyte in a biological sample using mass spectrometry
WO2007103734A2 (en) * 2006-03-03 2007-09-13 Ciphergen Biosystems, Inc. Method for quantifying an analyte in a biological sample using mass spectrometry
US20110076697A1 (en) * 2009-04-28 2011-03-31 Innovative Laboratory Technologies, Inc. Lateral-flow immuno-chromatographic assay devices
US8546148B2 (en) 2010-06-14 2013-10-01 Siemens Healthcare Diagnostics Inc Composition for use as an assay reagent
US11193929B2 (en) * 2014-12-18 2021-12-07 Biomerieux Synthetic bi-epitope compound
CN105628941A (zh) * 2015-12-22 2016-06-01 北京康彻思坦生物技术有限公司 一种IgM血清学检测质控物的制备方法
CN105628941B (zh) * 2015-12-22 2017-09-22 北京康彻思坦生物技术有限公司 一种IgM血清学检测质控物的制备方法

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